CN109576397B

CN109576397B - Human immunodeficiency virus type 1 nucleic acid quantitative detection kit

Info

Publication number: CN109576397B
Application number: CN201811619144.XA
Authority: CN
Inventors: 蒋析文; 夏乔; 李汉荣; 杨鸿辉; 廖丽丽; 王晶晶; 唐康明
Original assignee: Guangzhou Da'an Gene Co ltd
Current assignee: Guangzhou Da'an Gene Co ltd
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2022-10-14
Anticipated expiration: 2038-12-28
Also published as: CN109576397A

Abstract

The invention provides a quantitative detection kit for human immunodeficiency virus type 1 nucleic acid, in particular to a detection kit which utilizes a real-time fluorescence polymerase chain reaction technology to identify and quantify HIV-1RNA nucleic acid, and can be widely applied to multiple fields of window phase detection, clinical diagnosis, scientific research, curative effect tracking, inspection and quarantine, epidemic situation prevention and control and the like of AIDS caused by HIV virus.

Description

Human immunodeficiency virus type 1 nucleic acid quantitative detection kit

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a nucleic acid quantitative detection kit for human immunodeficiency virus type 1 (HIV-1).

Background

Human Immunodeficiency Virus (HIV), i.e., AIDS Virus, is a kind of retrovirus that is a Lentivirus (Lentivirus) that infects cells of the Human immune system. In 1981, human immunodeficiency virus was first discovered in the United states. Because the HIV virus directly attacks the human CD4-T lymphocyte system, the immune system is damaged irreversibly, and once the virus invades the cells of the body, the virus is integrated with the human cells and is difficult to eliminate all the year round. AIDS patients cannot be cured after being infected with other diseases, malignant tumors can occur, and the fatality rate is extremely high. In 2017, 10, 27 days, the national cancer research institute published the list of carcinogens for preliminary reference, and the human immunodeficiency virus type I (infection) is in a list of carcinogens.

AIDS can have a long asymptomatic latent period after infection, and HIV infected people further infect healthy people through sexual contact, blood contact and mother-to-infant transmission during the latent period, thereby bringing great health and safety threats to the society. In 2011, 250 thousands of HIV infection patients are newly added all over the world, and 170 thousands of HIV infection-related deaths are. The overall prevention and treatment form of HIV infection is still very severe, and the primary screening reagent for HIV detection with high quality and high cost performance has great market demand. The key to controlling the prevalence of AIDS is to enhance the HIV screening, early diagnosis and epidemic situation suppression of people, so the HIV detection method with high sensitivity and short window period has great significance to the control of HIV transmission.

AIDS patient samples are at extreme biohazard risk; detection is only possible under the highest level of biological protection. Aids virus infection can be clearly diagnosed by several types of laboratory tests, such as: antibody capture enzyme-linked immunosorbent assay (ELISA), antigen detection assay, serum neutralization assay, real time RT-PCR detection and virus isolation culture.

The serological neutralization test and the virus isolation culture have the defects of high safety risk, low sensitivity, immunological cross reaction, long period and the like, and are not suitable for the preliminary screening of people requiring high speed and high flux. The antigen-antibody detection method has the advantages of good sensitivity and rapid reaction, and has the obvious defects that the window period of 1-3 months is generated at the initial stage of the HIV infection, sufficient antibodies do not generate reaction in the serum of a patient during the window period, the antigen-antibody detection result is negative or uncertain, the early diagnosis of the HIV infection is not facilitated, the detection is missed, and the risk of HIV transmission is possibly caused; the antibody at the end of AIDS is also obviously reduced, which is not beneficial to detection and tracking. The sensitivity of common PCR is low, the HIV variation degree is very high, the number of popular subtypes is very large, and the detection effect is not ideal.

Therefore, there is an urgent need in the art to develop a method for HIV detection with high sensitivity and short window to meet clinical needs.

Disclosure of Invention

The invention aims to provide a human immunodeficiency virus type 1 (HIV-1) nucleic acid quantitative detection kit which is high in sensitivity and convenient to use.

In a first aspect of the invention, a PCR primer pair group for detecting human immunodeficiency virus type 1 (HIV-1) is provided, wherein the primer pair group comprises a first primer pair, and the first primer pair comprises a forward primer shown as SEQ ID NO. 1; and, a reverse primer as set forth in SEQ ID No. 2.

In another preferred example, the primer pair group further comprises a second primer pair, and the second primer pair comprises a forward primer shown as SEQ ID No. 4; a forward primer as set forth in SEQ ID No. 5; and, a reverse primer as shown in SEQ ID No. 6.

In a second aspect of the present invention, there is provided a probe set for detecting human immunodeficiency virus type 1, the probe set comprising: a probe specific to the conserved sequence of LTR gene as shown in SEQ ID NO. 3.

In another preferred embodiment, the probe set further comprises: a probe specific to GAG gene conserved sequences as shown in SEQ ID No. 7.

In a third aspect of the present invention, a kit for detecting human immunodeficiency virus type 1 is provided, the kit comprising an HIV-1 reaction solution a, the HIV-1 reaction solution a comprising a PCR primer pair group according to the first aspect of the present invention.

In another preferred embodiment, the HIV-1 reaction solution A further comprises a probe set according to the second aspect of the present invention.

In another preferred embodiment, the kit further comprises the HIV-1 reaction liquid B, and the HIV-1 reaction liquid B comprises one or more components selected from the following group:

hot start Taq enzyme, reverse transcriptase, and dNTPs.

In another preferred embodiment, the kit further comprises an HIV-1 quantitative reference.

In another preferred embodiment, the kit further comprises a negative quality control product.

In another preferred example, the kit also comprises an HIV-1 strong positive quality control product.

In another preferred embodiment, the kit also comprises an HIV-1 critical positive quality control product.

In another preferred embodiment, the kit further comprises an internal standard quality control product.

In another preferred embodiment, the kit further comprises a reverse transcription reagent.

In a fourth aspect of the present invention, there is provided a method for detecting human immunodeficiency virus type 1, the method comprising the steps of:

(1) Providing a sample to be detected, wherein the sample contains HIV-1RNA;

(2) Preparing an amplification reaction system, carrying out amplification reaction, drawing an amplification curve, and calculating a threshold value cycle value so as to obtain a quantitative detection result of the sample nucleic acid:

wherein, the amplification reaction system comprises the sample to be detected provided in step (1), a reverse transcription reagent for reverse transcription of HIV-1RNA in the sample to be detected into cDNA, the primer pair group of the first aspect of the invention, and the probe group of the second aspect of the invention.

In another preferred embodiment, the method is for non-diagnostic purposes.

In a fifth aspect, the present invention provides the use of the primer pair set of the first aspect, and the probe set of the second aspect, to prepare a detection kit for the quantification of human immunodeficiency virus type 1 (HIV-1) nucleic acids.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1:20 parts of sensitivity reference RT-PCR amplification curve;

FIG. 2: the RT-PCR amplification curves of the accurate reference products L0-L8;

FIG. 3: quantifying a standard curve for the linear range;

FIG. 4: 1.0X 10 ⁵ IU/ml precision reference amplification curve;

FIG. 5:1.0×10 ⁴ IU/ml precision reference substance amplification curve;

FIG. 6: 1.0X 10 ³ IU/ml precision reference amplification curve.

FIG. 7 amplification products for the LTR Gene singleplex System

FIG. 8 amplification product for GAG Gene singleplex System

FIG. 9 Dual amplification System results for LTR Gene and GAG Gene

FIG. 10 clinical examination results of control primer set 1

FIG. 11 clinical test results of control primer set 2

FIG. 12 control PCR primer set 3 single system for clinical plasma sample detection results

FIG. 13 control PCR primer set 3 multiplex assay versus clinical plasma sample assay results

FIG. 14 electrophoresis results of control PCR primer set 3 multiplex detection

Detailed Description

The invention belongs to the field of pathogen molecule detection, and relates to a high-sensitivity human immunodeficiency virus type 1 (HIV-1) RNA detection kit, which can be widely applied to multiple fields of window detection, clinical diagnosis, scientific research, curative effect tracking, inspection and quarantine, epidemic situation prevention and control and the like of AIDS caused by HIV viruses.

HIV virus nucleic acid in blood plasma exists in bodies of all infected persons, the high-sensitivity nucleic acid detection can greatly shorten the window period of antigen-antibody detection, the fluorescence PCR technology has the advantages of short amplification fragments, dual specificity of primers and probes and the like, multiple fluorescence PCR has multiple guarantees because multiple genes are detected simultaneously, the missing detection risk is greatly avoided, the multiple real-time fluorescence PCR technology is applied to the rapid detection of the AIDS virus, the detection and identification efficiency can be improved, and support and basis are provided for clinical rapid diagnosis and targeted treatment. Especially has great clinical application value for the self-test diagnosis of newborn (mother-baby transmission) suspected to be infected with HIV and asymptomatic suspected infected persons, and has great significance and great social benefit for the screening of blood products.

The multiplex real-time fluorescence PCR technology is an advanced technology combining the PCR technology and a multicolor fluorescence labeling probe, has the characteristics of rapidness, specificity, sensitivity, high automation degree and the like, is combined with anti-pollution technology processing, and is particularly suitable for the requirements of large-scale and rapid diagnosis. The technology adopts different fluorescent groups to mark specific probes, and can simultaneously amplify various viruses in the same PCR reaction tube by matching with a multiplex PCR technology, wherein the fluorescent growth signal and the growth quantity of a corresponding PCR product form an equal ratio relation and are collected by an automatic fluorescence detector, and finally the purpose of detecting the types of pathogens can be achieved by analyzing a fluorescent growth curve. Because the fluorescent PCR technology has the advantages of short amplified fragment, dual specificity of the primer and the probe and the like, the multiple real-time fluorescent PCR technology is applied to the rapid detection of the AIDS virus, the detection and identification efficiency can be improved, whether suspected cases or suspected media infect the AIDS virus or not and the type of clinic infection can be determined in a short time, necessary prevention and control and treatment measures can be conveniently and timely taken clinically, the viruses are prevented from being continuously transmitted, the diagnosis and treatment efficiency is improved, and support and basis are provided for rapid diagnosis, effective monitoring and targeted treatment.

Multiplex PCR (multiplex PCR), also called multiplex primer PCR or composite PCR, is a PCR reaction in which more than two pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments, and the reaction principle, reaction reagents and operation process are the same as those of general PCR.

There are many factors that affect multiplex PCR reactions, such as:

(1) The imbalance of the reaction system causes some dominant primers and templates thereof to be rapidly amplified in the early-stage several reactions, and a large amount of amplification products are obtained, and the amplification products are good inhibitors of DNA polymerase at the same time. Therefore, the polymerization ability of polymerase is more and more strongly inhibited with the occurrence of a large amount of amplification products, and thus, primers and templates thereof which are at a disadvantage in the early stage are more difficult to react, and finally, the amount of amplification products is so small that they cannot be detected.

(2) The primer specificity, if the primer has stronger binding force with other non-target gene fragments in the system, the ability of the target gene to bind the primer is contended, thereby leading to the reduction of the amplification efficiency.

(3) The optimal annealing temperatures are different, a plurality of pairs of primers are placed in a system for amplification, and the optimal annealing temperatures of each pair of primers are required to be close to each other because the annealing temperatures for PCR reaction are the same.

(4) Primer dimers, including dimers between primers and hairpin structures formed by the primers themselves, are third-party DNA-mediated dimers, and these dimers, like non-specific primers, interfere with the competition between primers and target binding sites, affecting amplification efficiency.

Although several factors affecting amplification efficiency are mentioned above, more are not yet clear. To date, there is no effective method for clearly predicting amplification efficiency.

In a preferred embodiment of the invention, a scheme for quantitatively detecting HIV-1RNA by a fluorescent quantitative PCR method is provided, and particularly relates to a kit for detecting and quantifying HIV-1RNA nucleic acid by a real-time fluorescent PCR technology with high sensitivity.

The invention adopts the following technical scheme:

(1) Specific primers and probes designed for HIV-1 highly conserved region

(2) Obtaining cDNA by reverse transcription of sample RNA by one-step method

(3) Specific amplification of two conserved sequences of HIV by single-tube multiplex PCR reaction

(4) And automatically drawing an amplification curve and calculating a threshold value cycle value by an instrument to obtain a quantitative detection result of the sample nucleic acid.

(5) The validity of the detection result is ensured by the quality control of the positive control quality control product, the quantitative quality control product, the negative quality control product and the internal standard quality control product.

The specific embodiment of the invention is as follows:

in the pair GENBANK and International HIOn the basis of comparison of the known HIV-1 nucleic acid sequences in the V database, specific conserved regions of the nucleic acid sequences of various subtypes are respectively searched, and two target nucleotide probes and corresponding primers are respectively designed on two gene conserved regions of an LTR region and a GAG region which are far away from each other. The primer probes are applied to an RT-PCR reaction enzyme system containing heat-resistant DNA polymerase, reverse transcriptase and high-quality deoxyribonucleoside triphosphates (dNTPs) and a reaction system containing Mg ²⁺ In the RT-PCR reaction solution with equal components, the circulating amplification of in vitro nucleic acid is realized by a fluorescence PCR instrument.

According to the embodiment of the invention, the kit comprises the following main components: HIV-1 reaction liquid A, HIV-1 reaction liquid B, HIV-1 quantitative reference substance, negative quality control substance, and HIV-1 strong positive quality control substance (content 1.0 × 10) ⁵ IU/ml), HIV-1 critical positive quality control (content 1.0X 10) ³ IU/ml) and internal standard quality control (content 1.0 × 10) ³ IU/ml); the HIV-1 quantitative reference comprises: HIV-1 quantitative reference 1 (1.0X 10) ⁶ IU/ml); HIV-1 quantitative reference 2 (1.0X 10) ⁵ IU/ml); HIV-1 quantitative reference 3 (1.0X 10) ⁴ IU/ml); HIV-1 quantitative reference 4 (1.0X 10) ³ IU/ml)。

According to an embodiment of the present invention, the primer probe mixture consists of the following 5 HIV-specific forward and reverse primers and the following 2 specific probes.

According to an embodiment of the present invention, the sequences of the forward and reverse primers specific for the conserved sequence of the HIV-1 type LTR gene are 5; the sequence of the probe specific to the conserved sequence of HIV-1 LTR gene is 5.

According to an embodiment of the present invention, the sequences of the forward and reverse primers specific for the conserved sequence of the HIV-type 1 GAG gene are 5; 5 'TCTTTGGGACAGACCCCTCGTT-doped 3' (SEQ ID NO: 5) and 5 'CTCCAATTCCCCTCTATCATTT-doped 3' (SEQ ID NO: 6); the sequence of the specific probe aiming at the conserved sequence of HIV-type 1 GAG gene is 5' TTTCCATCTTCCTGGCAAATTCATT-3' (SEQ ID NO: 7), and the two ends of the probe are respectively combined with a fluorescence generating group FAM and a fluorescence quenching group 3' eclipse +5Zip.

In a preferred embodiment of the invention, the primer concentration in the HIV-1 reaction solution A is 0.1-0.2mmol/L, and the probe concentration is 0.04-0.1mmol/L.

In another preferred embodiment of the present invention, the HIV-1 reaction solution A contains Tris-HCl (50 mmol/L, pH 8.0), mgCl ₂ (2-8mmol/L)、KCl(50-100mmol/L)。

In another preferred embodiment of the present invention, HIV-1 reaction solution B is composed of hot start Taq enzyme, reverse transcriptase, dNTPs. The reverse transcriptase can be selected from conventional reverse transcriptases such as mMLV. The hot start Taq enzyme, the reverse transcriptase and the dNTPs can be commercially available products, such as products of Promega company, wherein the dosage of the hot start Taq enzyme in each HIV-1 reaction liquid B is 3-10U, the dosage of the reverse transcriptase is 2-4U, and the dosage of the dNTPs is 100-500 mu mol.

According to the embodiment of the invention, HIV-1 reaction liquid A and HIV-1 reaction liquid B are mixed into reaction liquid mixture with the total volume of 20uL according to the volume of 17 uL and 3 uL for detecting each single-person PCR reaction liquid, 40uL of nucleic acid of a sample to be detected is added, and then real-time fluorescent quantitative PCR reaction is carried out.

Another preferred embodiment of the present invention is that the conditions for PCR amplification are: reverse transcription at 50 deg.c for 15 min for 1 cycle; step (2), performing pre-denaturation at 95 ℃ for 15 minutes for 1 cycle; step (3), denaturation-annealing is carried out for 94 ℃,15 seconds, then 55 ℃ and 45 seconds, and 45 cycles are carried out; the collection of the fluorescence signal is performed after 45 seconds at 55 ℃ for each cycle of step (3).

According to the embodiment of the invention, the kit also provides quality control products which are respectively a negative quality control product, an HIV-1 strong positive quality control product, an HIV-1 critical positive quality control product and an HIV-1 quantitative reference product 1 (1.0 multiplied by 10) ⁶ IU/ml); HIV-1 quantitative reference 2 (1.0X 10) ⁵ IU/ml); HIV-1 quantitative reference 3 (1.0X 10) ⁴ IU/ml); HIV-1 quantitative reference 4 (1.0X 10) ³ IU/ml) and an internal standard quality control product. The negative quality control product is a detected HIV-1 antibodyNegative, HIV-1RNA PCR detection of negative healthy human plasma. The positive quality control product and the quantitative reference product are artificially synthesized virus-like particles, the virus-like particles contain HIV-1 target gene segments amplified by the primers designed by the invention, and the selection concentrations are as follows: 1.0X 10 ⁶ IU/mL、1.0×10 ⁵ IU/mL、1.0×10 ⁴ IU/mL、1.0×10 ³ IU/mL of RNA virus-like particles are prepared into quantitative reference substances. When the test is carried out on the sample to be tested in the kit, a negative quality control product, a positive quality control product and a quantitative reference product must be extracted at the same time, and only when the positive quality control product is detected, the fluorescence signals of the FAM channel are all positive, and the fluorescence signals of the negative quality control product are all negative, and when the logarithm increase period exists in the VIC detection channel, the detection result of the sample to be tested is effective.

The reaction liquid of the kit only contains 2 reaction liquids of the liquid A and the liquid B, and can be conveniently matched with an automatic pipettor to be used in a large number of configurations. The amplification of the sample to be detected by the kit is automatically completed by a commercially available fluorescence quantitative PCR instrument, the operation is simple, the time consumption is low, and the occurrence of subsequent pollution is reduced to the maximum extent. The detection result can be used for research in multiple fields such as HIV-1 rapid screening, auxiliary clinical diagnosis, routine monitoring and the like.

According to an embodiment of the invention, the primer probe mixture consists of 5 HIV-specific forward and reverse primers and 2 specific probes.

The amplification of the specimen to be detected by the kit is automatically completed by a commercially available fluorescence quantitative PCR instrument, the operation is simple, the time consumption is low, and the pollution is reduced to the maximum extent. The detection result can be used for multiple field researches such as HIV virus rapid diagnosis, auxiliary clinical research, routine monitoring and the like.

The main advantages of the invention are:

(1) The kit can be used for carrying out amplification detection on nucleic acids of different subtypes of HIV-1 viruses, can reflect the state of HIV-1 virus infection in a sample, greatly shortens the window period of detection compared with antibody reaction detection, is beneficial to early diagnosis and treatment of the HIV-1 viruses, and has great significance for monitoring, prevention and control of the HIV-1 viruses and clinical diagnosis of AIDS;

(2) Specific primers and probes are designed respectively aiming at the nucleic acid conserved sequences of different subtypes such as the A-H subtype of the HIV-1 virus M group, the epidemic recombinant CRF-AE type and the like, so that the specificity and the accuracy of detection are ensured, the advantages of wide coverage, dual target point guarantee and the like are also realized, and the reliability of a detection result is ensured;

(3) Only 1.5-2h is needed for one detection process of one sample, and compared with the existing detection technology of a nucleic acid hybridization method, the time required by detection is shortened by more than half;

(4) The reagent adopts virus-like particles which are artificially synthesized in vitro and contain HIV-1RNA as a positive quality control product, namely the reagent can be subjected to quality control, has higher effectiveness and scientificity than plasmid as the quality control, and simultaneously greatly improves the safety of operators;

(5) The kit is provided with a negative quality control product, a positive quality control product and an internal standard substance, and can be used for monitoring the whole process of sample nucleic acid extraction and PCR amplification so as to reduce the occurrence of false negative;

(6) The reagent adopts a large packaging device of RT-PCR reaction liquid, primer probe mixed liquid and an RT-PCR reaction enzyme system, is suitable for various fluorescent detection devices, and has the characteristic of wider applicability.

(7) The method of the invention needs few samples, has stable and efficient performance and high accuracy.

The present invention will be described in further detail with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1: human immunodeficiency virus type 1 (HIV-1) nucleic acid quantitative detection kit (PCR-fluorescent probe method)

The kit applies a fluorescent quantitative PCR method to quantitatively detect the human immunodeficiency virus type 1 nucleic acid (HIV-1) RNA in a serum or plasma sample, and is suitable for the auxiliary diagnosis of the human immunodeficiency virus type 1 infection and the monitoring of the curative effect of the drug treatment of the human immunodeficiency virus type 1 infected person.

The kit comprises the following reagents:

the specific primer probe sequence is as above.

And (3) a test scheme:

1. sample processing and nucleic acid extraction (sample processing zone)

The negative quality control material, the positive quality control material and the quantitative reference material in the kit all participate in extraction and are used for monitoring the environment and controlling the quality of a PCR detection reagent. The internal standard solution in the kit participates in the extraction process, so the kit needs to be matched with an extraction reagent for use, and the recommended use amount is 5 mul of the internal standard solution added into each sample to be extracted for quality control of the extraction process. The positive quality control material and the negative quality control material are regarded as having infectious substances, and the operation and the treatment are required to meet the requirements of relevant regulations.

PCR reagent preparation (reagent preparation area)

The reagent with large package specification is applicable:

taking the HIV-1 reaction solution A and the HIV-1 reaction solution B out of the kit, melting at room temperature, shaking, mixing uniformly, and centrifuging at 8,000rpm for several seconds for use.

Taking N (N = the number of samples to be detected +1 negative quality control product +1 strong positive quality control product +1 critical positive quality control product +4 positive quantitative reference products) PCR reaction tubes, and preparing an HIV-1 single-person amplification system as follows:

components	HIV-1 reaction solution A	HIV-1 reaction solution B	Total volume
				Dosage of	15μl	5μl	20μl

The components are fully mixed, short-time centrifugation is carried out after mixing, all liquid on the tube wall is centrifuged to the tube bottom, and then 20 mul of amplification system is subpackaged into a PCR tube.

3. Application of sample (sample preparation zone)

And respectively adding 40 mu l of the extracted sample to be tested, the negative quality control product, the HIV-1 strong positive quality control product, the HIV-1 critical positive quality control product and the HIV-1 quantitative reference product nucleic acid into the HIV-1 reaction tube by using a suction nozzle with a filter element. The tube caps were closed and centrifuged at 8,000rpm for several seconds before transferring to the amplification detection zone.

PCR amplification (amplification and product analysis region)

The reaction tube is placed in the sample tank of the instrument. ABI Prism 7500 instrument setup (ABI Prism 7300 instrument refers to this procedure, see description for details). Opening a 'Setup' window, setting negative quality control (NTC), positive quality control, unknown Sample (Unknow) and positive quantitative reference substance (Standard) according to the corresponding sequence of the samples, and setting Sample names in a column of 'Sample Name'; the probe detection mode is set as follows: reporter Dye1: FAM, quencher Dye1: TAMRA, reporter Dye2: VIC, quencher Dye2: none, passive Reference: NONE. Opening an instrument window, and setting the circulation conditions as follows:

15 minutes at 50 ℃ for 1 cycle;

15 minutes at 95 ℃ for 1 cycle;

94 ℃ for 15 seconds → 55 ℃ for 45 seconds (fluorescence collected), 45 cycles.

And after the setting is finished, saving the file and operating the program.

5. Analysis of results

And automatically storing the result after the reaction is finished, adjusting the Start Value, the End Value and the Threshold Value of Baseline according to the analyzed image (the user can automatically adjust according to the actual situation, the Start Value can be 3-15, the End Value can be 5-20, setting the Value of Threshold in a Log map window, enabling a base line to be positioned in the exponential phase of an amplification curve, adjusting the amplification curve of a negative quality control product to be straight or lower than a Threshold line), clicking Analysis to automatically obtain the Analysis result, viewing the result in a Report interface, and recording the Value (C) of an unknown sample.

6. Quality control

Negative quality control product: the FAM detection path amplification curve has no logarithmic growth period, and the VIC detection path amplification curve is a logarithmic growth period; HIV-1 positive quality control: the amplification curve of the FAM detection pathway has an obvious logarithmic growth period and is a typical S-shaped amplification curve, the requirements are simultaneously met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.

7. Result judgment

7.1 if FAM detection channel has no log-increase period and VIC detection channel has log-increase period, then the HIV-1RNA concentration of the sample is judged to be less than the detection sensitivity of the kit.

7.2 if the amplification of the FAM fluorescence signal is obvious, the amplification curve has obvious logarithmic growth period and is a typical S-shaped curve, and the Ct value is less than 45, judging according to the following method:

if the concentration of the sample is 50 ≦ C ≦ 1.00E +009, then the HIV-1RNA concentration of the sample = C IU/ml;

if the concentration of the sample is C > 1.00E +009, the HIV-1RNA concentration of the sample is > 1.0X 10 ⁹ IU/ml. If accurate quantification is required, the sample can be diluted to a linear range with a negative control and then tested. The HIV-1RNA concentration of the sample = (C × dilution multiple) IU/ml;

if the concentration of the sample is more than or equal to 20 and less than 50, and the amplification curve of the VIC detection channel has logarithmic growth period, the HIV-1RNA of the sample is positive, and the concentration is lower than the lower limit of the quantification of the kit and is only used as reference;

if the concentration of the sample is C < 20.0IU/ml and the amplification curve of the VIC detection channel has a logarithmic growth period, the HIV-1RNA of the sample is positive, the concentration is lower than the lower detection limit of the kit, and the concentration is only used as reference.

8. Interpretation of results

8.1. The negative quality control substances, the HIV-1 strong positive quality control substances and the HIV-1 critical positive quality control substances are required to be detected in each experiment, and the detection result can be judged when the quality control substance result meets the quality control requirement.

8.2. Positive result determination criteria: the amplification curves have logarithmic growth periods in both FAM and VIC detection channels; or the amplification curve has a logarithmic growth period in the FAM detection channel and has no logarithmic growth period in the VIC detection channel.

8.3. Negative result judgment criteria: the amplification curve has no obvious logarithmic growth period in FAM detection channel and has logarithmic growth period in VIC detection channel.

8.4. Report format:

the negative results report format was: human immunodeficiency virus type 1RNA is not detected in the sample, and the concentration is lower than the sensitivity of the kit;

the positive results report format was:

1) When the sample test results show that the concentration of HIV-1RNA is 50. Ltoreq. C.ltoreq.1.00E +009, the report format is as follows: human immunodeficiency virus type 1RNA is detected in the sample, and the concentration of the RNA is C IU/ml;

2) When the test results for the sample are HIV-1RNA concentrations C > 1.00E +009, the report format is: the concentration of the RNA of the human immunodeficiency virus type 1 detected by the sample is more than 1.0 multiplied by 10 ⁹ IU/ml, if detected after dilution, the report format is: human immunodeficiency virus type 1RNA is detected in the sample, and the concentration is (C multiplied by dilution) IU/ml.

3) If the C is more than or equal to 20IU/ml and less than 50IU/ml of the sample, and the amplification curve of the VIC detection channel has a logarithmic growth period, the report format is as follows: the sample has low human immunodeficiency virus type 1RNA load, and the measured value is only used for reference;

4) When the test result of the sample has HIV-1RNA concentration C less than 20 and the amplification curve of the VIC detection channel has logarithmic growth period, the report format is as follows: the content of the human immunodeficiency virus type 1RNA is lower than the detection lower limit of the kit; if the internal standard detection channel amplification curve has no logarithmic growth period or no Ct value, the detection result of the sample is invalid, the reason should be searched and eliminated, and the sample is repeatedly detected.

Example 2: sensitivity detection assay

Sensitivity reference sample is prepared from 5 × 10 nominal concentration ⁵ IU/ml national reference (China food and drug testing institute), and 20 parts by weight of HIV-1 negative plasma diluted national reference to 20IU/ml serving as a sensitivity reference. And simultaneously extracting nucleic acid from 20 parts of sensitivity reference substances, a negative quality control substance, an HIV-1 strong positive quality control substance, an HIV-1 critical positive quality control substance and 4 positive quantitative reference substances.

The experiment is repeated for 3 times in parallel to obtain the same detection result, which shows that the primer of the invention has better specificity, the system has good amplification efficiency, the sensitivity detection is good, the sensitivity of the kit of the invention is 20IU/ml, the positive detection rate of 3 times of experiments reaches 100%, the positive quality control detection result is positive, and the negative quality control detection result is negative.

Example 3: accuracy testing experiment

The accuracy reference sample is artificially synthesized RNA virus-like particles with the concentration calibrated by national reference, and HIV-1 negative plasma is diluted to be 1.0 multiplied by 10 respectively ⁹ IU/ml、1.0×10 ⁸ IU/ml、1.0×10 ⁷ IU/ml、 1.0×10 ⁶ IU/ml、1.0×10 ⁵ IU/ml、1.0×10 ⁴ IU/ml、1.0×10 ³ IU/ml、1.0×10 ² IU/ml and 50IU/ml, which are used as accuracy reference substances and are respectively numbered as L0-L8 in 9 parts. And simultaneously extracting nucleic acid from 9 parts of the accuracy reference substance, the negative quality control substance, the HIV-1 strong positive quality control substance, the HIV-1 critical positive quality control substance and 4 positive quantitative reference substances.

According to the steps in example 1, HIV-1 reaction liquid A and HIV-1 reaction liquid B are taken out from the kit, 16 PCR reaction systems (9 samples to be tested with accuracy, 1 negative quality control product, 1 strong positive quality control product, 1 critical positive quality control product and 4 positive quantitative reference products) are prepared in total by conversion according to the proportion of a single part, and then 20 microliter of amplification system of each part is subpackaged into PCR tubes. Adding 40 μ l of extracted accuracy reference substance, negative quality control substance, HIV-1 strong positive quality control substance, HIV-1 critical positive quality control substance and HIV-1 quantitative reference substance nucleic acid into the HIV-1 reaction tube. The tube caps were closed, centrifuged at 8,000rpm for several seconds and transferred to an amplification detection zone. According to the PCR amplification procedure in example 1, 9 accurate samples with known concentrations were evaluated using 4 standard curves for quantitative reference samples, and the results of theoretical concentration and detection concentration were compared. The results are shown in the following table.

TABLE 1 accuracy reference experiment results

Experimental results show that the fixed value results of 9 parts of the accuracy reference products L0-L8 are not more than the true value +/-0.5 Log value, the quality is controllable, and the fixed value results are qualified. 50 to 1.0X 10 for viral load ⁹ The virus samples in IU/ml range can be effectively detected in a fixed value by using the human immunodeficiency virus type 1 (HIV-1) nucleic acid quantitative detection kit, and the error range does not exceed 0.5Log 10 logarithm values. The kit has the advantages that the quality control product controls the effectiveness of each fixed value experiment, the experimental error is avoided, the deviated virus load fixed value result is obtained, and the result is controllable and effective. Meanwhile, the PCR system of the kit has a wider quantitative linear range and high accuracy, and can meet different clinical detection requirements.

Example 4: precision detection experiment

The precision reference sample is artificially synthesized RNA virus-like particles with the concentration calibrated by national reference substances, and the dilution of HIV-1 negative plasma is 1.0 multiplied by 10 respectively ⁵ IU/ml、1.0×10 ⁴ IU/ml、1.0×10 ³ 10 parts of IU/ml are used as precision reference substances, and the total amount is 30 parts. And simultaneously carrying out nucleic acid extraction on 30 parts of precision reference substances, a negative quality control substance, an HIV-1 strong positive quality control substance, an HIV-1 critical positive quality control substance and 4 positive quantitative reference substances.

According to the steps in example 1, HIV-1 reaction liquid A and HIV-1 reaction liquid B are taken out from the kit, 37 parts (30 samples to be measured with precision +1 negative quality control product +1 strong positive quality control product +1 critical positive quality control product +4 positive quantitative reference products) of PCR reaction systems are prepared in a total manner by conversion of a single-person proportion, and then 20 mul of amplification systems of each person are subpackaged in PCR tubes. Adding 40 μ l of the extracted precision reference substance, negative quality control substance, HIV-1 strong positive quality control substance, HIV-1 critical positive quality control substance and HIV-1 quantitative reference substance nucleic acid into the HIV-1 reaction tube. The tube caps were closed and centrifuged at 8,000rpm for several seconds before transferring to the amplification detection zone. According to the PCR amplification procedure in example 1, the precision-based samples were subjected to the calibration using 4 calibration curves for quantitative reference samples, and the coefficient of variation CV of the LOG value of the calibration concentration was calculated. The results are shown in the following table.

TABLE 2 precision reference experimental results

The results show that the quantitative reference value is 1.0X 10 ⁵ IU/ml、1.0×10 ⁴ IU/ml、1.0×10 ³ IU/ml is respectively set to 10 precision reference substances, and the variation coefficient of the concentration logarithm values of the precision reference substances with three different concentrations is less than 5 percent, which shows that the method has consistent CT value for the sample set value of the same virus load, small standard deviation and good stability and precision evaluation of a plurality of measurement results.

Example 5: electrophoretic detection of amplification products

The present inventors designed PCR amplification primers for HIV-1 type LTR gene and GAG gene, and optimized each component of PCR system to obtain good amplification product. The embodiment utilizes a single-fold system optimized for LTR gene design, a single-fold system optimized for GAG gene design and a double-amplification system for LTR gene and GAG gene to respectively carry out PCR amplification on clinical plasma samples and detect the amplification condition of the system products through electrophoresis.

The specific detection steps and detection conditions are the same as those in the above example, the result of a single system designed by using LTR genes is shown in FIG. 7, the leftmost lane in FIG. 7 is Marker, the rest lanes are 5 different clinical plasma samples, all the samples are positive to the target genes, and the product band is single and bright.

The result of the single system designed by using GAG gene is shown in figure 8, the leftmost lane in figure 8 is Marker, the other lanes are 5 different clinical plasma samples, the samples are all positive to the target gene, and the product band is single and bright.

The results of the dual amplification system using LTR gene and GAG gene are shown in FIG. 9, the leftmost lane in FIG. 9 is Marker, the rest lanes are 10 different clinical plasma samples and 1 negative plasma, the samples are all positive to the target gene, the product band is single bright, and the negative sample has no product.

The detection result shows that the primer and the amplification system of the invention are optimized, the target gene of the clinical sample is normally amplified, and the specificity of the amplified product are good.

Comparative example 1

The inventor designs ten pairs of PCR amplification primers aiming at HIV-1 type LTR genes and GAG genes, wherein most of the primers have low sensitivity and poor specificity, have missing detection and can not meet the detection requirement, and typical common PCR primer sequences and detection effect data are as follows:

control primer pair 1 (for conserved sequence of HIV-1 type LTR gene)

The upstream primer is CCCGCTTAAATACTGACGC (SEQ ID NO.: 8);

downstream primer GCACGGCAAGAGGCGA (SEQ ID No.: 9)

Control RT-PCR primer pair 2 (conserved sequence against HIV-type 1 GAG gene):

the upstream primer is ACCTCCAATTCCCTCAC (SEQ ID NO.: 10);

the downstream primer is TGGCAACGACCCCTC (SEQ ID NO: 11).

The specific detection steps and detection conditions are the same as those in the above embodiment, the detection result of the control primer pair 1 is shown in fig. 10, 10 different clinical plasma samples, 3 of which are positive for the target gene and 7 of which are negative for detection, indicate that the sensitivity of the control primer pair 1 is poor and the omission condition is serious.

The detection results using the control primer pair 2 are shown in fig. 11, and 10 different clinical plasma samples, of which 5 are positive for the target gene and 5 are negative for detection, indicate that the control primer pair 2 cannot detect the target gene in part of the samples, and have poor sensitivity and poor specificity.

Comparative example 2

To meet the requirement of multiplex detection, the present inventors performed a combination test on the selected pairs of PCR primers, wherein the vast majority of the combinations could not meet the requirement of multiplex detection, such as multiplex detection using a combination of the control primer pair 3 and the specific primers of the present invention against the conserved sequence of HIV-1 type GAG gene.

Control PCR primer pair 3 (conserved sequence for HIV-1 type LTR gene):

the upstream primer is CCGCTTAATACTGACGC (SEQ ID NO.: 12);

the downstream primer is CACGGCAAGAGGCGA (SEQ ID NO.: 13).

The detection steps and detection conditions are the same as the above embodiment, wherein the control PCR primer pair 3 is used for detecting the LTR gene conserved sequence, the result is shown in FIG. 12, 10 different clinical plasma samples are amplified, 10 target genes are positive, and the surface system has good specificity and sensitivity.

Multiplex detection results:

the results of the fluorescent PCR assay for multiplex detection are shown in FIG. 13. The electrophoresis product result is shown in fig. 14, the rightmost lane is Marker, the other lanes are clinical samples, no target band exists, and large non-specific amplification products exist, which indicates that the two sets of primer pairs have serious mutual influence and cannot perform multiplex detection.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Claims

1. A kit for detecting human immunodeficiency virus type 1 is characterized by comprising HIV-1 reaction liquid A, wherein the HIV-1 reaction liquid A comprises a PCR primer pair group and a probe group;

wherein the primer pair group comprises a first primer pair and a second primer pair:

the first primer pair comprises a forward primer shown as SEQ ID NO.1 and a reverse primer shown as SEQ ID NO. 2;

the second primer pair comprises a forward primer shown as SEQ ID NO.4, a forward primer shown as SEQ ID NO.5 and a reverse primer shown as SEQ ID NO. 6;

the probe set comprises:

a specific probe for the conserved sequence of LTR gene as shown in SEQ ID NO.3 and a specific probe for the conserved sequence of GAG gene as shown in SEQ ID NO. 7.

2. The kit of claim 1, wherein the kit further comprises an HIV-1 quantitative reference.

3. The kit of claim 1, wherein the kit further comprises a negative quality control.

4. The kit of claim 1, wherein the kit further comprises an HIV-1 strong positive quality control.

5. The kit of claim 1, wherein the kit further comprises an HIV-1 critically positive quality control.

6. The kit of claim 1, further comprising an internal standard quality control.

7. The kit of claim 1, wherein the kit further comprises a reverse transcription reagent.

8. The kit of claim 1, further comprising an HIV-1 reaction solution B, wherein the HIV-1 reaction solution B comprises one or more components selected from the group consisting of:

hot start Taq enzyme, reverse transcriptase and dNTPs.

9. A method for detecting human immunodeficiency virus type 1 for non-diagnostic purposes, said method comprising the steps of:

(1) Providing a sample to be detected, wherein the sample contains RNA of HIV-1;

the amplification reaction system comprises the sample to be detected, a reverse transcription reagent for reverse transcribing RNA of HIV-1 in the sample to be detected into cDNA, a primer pair group and a probe group, wherein the sample to be detected is provided in the step (1);

the probe set comprises:

a probe aiming at the specificity of the conserved sequence of the LTR gene shown in SEQ ID NO.3 and a probe aiming at the specificity of the conserved sequence of the GAG gene shown in SEQ ID NO. 7.